Vitamin B12 (cobalamin, Cbl) is an essential cofactor in human (and mammalian) metabolism for the enzymatic methylation of homocysteine in the biosynthesis of methionine, and for the molecular rearrangement of methylmalonyl-CoA to succinyl-CoA in human (and animal) cellular metabolism. A deficiency of this vitamin results in severe megaloblastic anemia and neuropsychiatric disorders. Two integrated physiological processes are required for sufficient Cbl to be provided to tissues. The first is intestinal absorption, and this process is mediated by gastric intrinsic factor (IF) which complexes with Cbl in the upper small intestine and transports it as IF-Cbl to the distal ileum where it binds to an IF receptor on the microvillus membranes of the epithelial mucosa and is internalized by a process of endocytosis. Within the ileal mucosal cell, the released Cbl moves unidirectionally to the portal blood where it appears bound to the plasma transport protein, transcobalamin II (TCII). TCII mediates the second essential physiologic process, which is transport of the vitamin in the plasma, as the TCII-Cbl complex, to the tissues where it binds to a specific TCII receptor on the plasma membrane of cells and is then internalized by receptor mediated endocytosis. Perturbations of this complex process that lead to Cbl deficiency result from impaired intestinal absorption (lack of IF or intestinal malabsorption) and lack of TCII. Accordingly, the long-term objectives and aims of this project are: i) to study the regulation of expression of TCII; ii) to characterize the congenital disorders of TCII deficiency; iii) to elucidate the integrated function of TCII and IF in facilitating the intestinal absorption of Cbl. To achieve these aims, we will characterize the genomic cis elements of the TCII gene (which we have cloned) and the cytoplasmic trans-active factors that regulate constitutive and tissue specific expression of TCII, and identify the mutations of the TCII gene that are responsible for the three congenital forms of abnormal TCII expression. These genetic studies of TCII should identify the factors that up-regulate synthesis of TCII in autoimmune diseases, B-cell proliferations and some cancers, and which may also down-regulate TCII synthesis as observed in some subjects. To achieve the third objective we will clone the IF receptor expressed on the ileal mucosa of guinea pig ileum. We will first purify analytical amounts of the receptor by affinity chromatography using an IF-Cbl affinity matrix and then obtain an amino acid sequence from the N-terminus and internal peptides and use the derived oligonucleotides to screen a cDNA library prepared from the mRNA of guinea pig ileal mucosa. The cDNA will provide information about the primary structure of the receptor, the transmembrane domain(s) and function, and will be useful to clone and characterize the IF-receptor gene. Such studies of the IF-receptor and TCII at the molecular level can clarify the role of TCII in the assimilation of Cbl and clarify the etiology of impaired Cbl absorption that can result in Cbl deficiency in the absence of deficiency of intrinsic factor.
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